Dr. Thomas Selby


Studies of transcription factors have provided important contributions toward understanding the cell morphology and developmental regulation of fruit flies based on sequence analysis of amino acid substitutions. In this report we describe the construction of four protein-DNA models based on two experimentally determined X-ray crystal structures (2DRP and 1A1H) using computational simulations of Z1, Z2, Z3, and Z4 zinc-finger DNA binding proteins. Once these models were constructed, 3 mutants were generated for the Z1/Z3 proteins. We evaluated the construction of the models and mutants against the templates using molecular mechanics (MM3) calculations in an attempt to determine the effect of the mutation at the molecular level. Our findings demonstrate that the Z1 mutant is greatly affected by the substitution of an arginine for a cysteine at position 57, and may lose function by binding DNA too tightly. However, the interaction energy of the Z3 mutants varies, with leucine substitution at position 55, not being critical for DNA binding, but a change at position 58 leucine to methionine having a moderate effect. These results also show that other factors including protein stability, degradation, and/or protein-protein interactions may be involved in the eventual morphological changes. The Z2/Z4 models were used as internal standards for our modeling analysis and demonstrated that the level of homology does not correlate with interaction energy values. These studies demonstrate the utility of structure-based analysis involving three-dimensional protein model building and molecular mechanics to evaluate amino acid substitutions that lead to changes in cellular processes. Additionally, this type of analysis allows the classification of amino acid substitutions into two groups: those that involve the loss of interaction energy and those that may have a loss of function due to other factors.



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